*Target (protein/gene name): ATP-dependent zinc metalloprotease/ FtsH *NCBI Gene # or RefSeq#: 1392 *Protein ID (NP or XP #) or Wolbachia#: *Organism (including strain): Bacillus Anthracis Etiologic Risk Group (see link below): Risk Group Lv 3 (high individual risk, low community risk)
*/Disease Information (sort of like the Intro to your Mini Research Write up): Anthrax is a serious infectious disease caused by gram-positive, rod-shaped bacteria known as Bacillus anthracis. Anthrax can be found naturally in soil and commonly affects domestic and wild animals around the world. The bacterial cell lives as a hardy spore to survive harsh conditions. The spores germinate into thriving colonies of bacteria once inside an animal or person. Anthrax usually affects livestock far more than humans, but—as we know from the 2001 anthrax attacks in the United States—anthrax is feared as a modern biological weapon. Anthrax occurs in three forms: 1.Cutaneous (affecting the skin) 2.Inhalational (in the lungs) Gastrointestinal (in the digestive tract)
Link to TDR Targets page (if present): Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) http://www.ncbi.nlm.nih.gov/gene/1086600 Essentiality of this protein: Targets mitogen-activated protein kinase cleaving them to remove a small N-terminal stretch but leaving the bulk of the protein, including the protein kinase domain, intact. LF-mediated cleavage of MEK1 and MKK6 has been shown to inhibit signalling through their cognate MAPK pathways
Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): multimer Complex of proteins?: Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
-- Show screenshot of BRENDA enzyme mechanism schematic
Reaction catalyzed by peptide-transporting ATPase
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): -- link to Sigma (or other company) page for assay (see Sigma links below) -- -or link (or citation) to paper that contains assay information -- links to assay reagents (substrates) pages. --- List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: -- For Homology Model option: ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB ---- Query Coverage: ---- Max % Identities: ---- % Positives ---- Chain used for homology:
Current Inhibitors: tissue inhibitors of metalloproteinases (TIMP1,2,3,4) Expression Information (has it been expressed in bacterial cells): E.coli Purification Method:
Amino acids 147–610 of FtsH from T. maritima were amplified from genomic DNA and ligated into a pET28a expression vector (Stratagene) adding a C-terminal hexa-His-tag. Expression in Rosetta (DE3) cells (Novagen) was induced by adding 0.5 mM IPTG at an OD600 of 0.5. Heat precipitation of E. coli proteins was carried out at 75°C for 3 min. The supernatant was further purified by Ni-NTA and anion-exchange chromatography. (Δtm)FtsH-containing fractions were again pooled and loaded onto an S75 size-exclusion column (Amersham Pharmacia Biosciences) equilibrated with buffer G (100 mM NaCl/2 mM DTT/20 mM Tris, pH 8.0/0.02% sodium azide). http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1413944/ Image of protein (PyMol with features delineated and shown separately):
The molecular architecture of the metalloprotease FtsH
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MNRIFRNTIF YLLIFLVVIG IVSYFNGSTQ KTTSVSYDKF ITKLEKGEVR NVQLQPKNGV FEVKGQFNNS SQGEQFVTYA PNTEELQKKI NDKAQGAEVK YQPAEETSAW VTFFTSIIPF VIIFILFFFL LNQAQGGGSR VMNFGKSKAK LYNDEKKKVR FRDVAGADEE KQELVEVVEF LKDPRKFAEV GARIPKGVLL VGPPGTGKTL LARAVAGEAG VPFFSISGSD FVEMFVGVGA SRVRDLFENA KKNAPCIIFI DEIDAVGRQR GAGLGGGHDE REQTLNQLLV EMDGFGANEG IIIIAATNRP DILDPALLRP GRFDRQITVD RPDVNGREAV LKVHARNKPL DENINLRAIA TRTPGFSGAD LENLLNEAAL VAARQDKKKI DMSDIDEATD RVIAGPAKKS RVISEKERNI VAFHEAGHTV IGVVLDEADV VHKVTIVPRG QAGGYAVMLP KEDRYFMTKP ELLDKITGLL GGRVAEEIVF GEVSTGAHND FQRATGIARR MVTEFGMSDK LGPMQFGSSQ GGQVFLGRDF HSEQNYSDAI AHEIDMEMQT IMKECYARAK QILTDNRDKL DLIAKTLLEV ETLDAEQINH LCDYGRLPER PTSSADVKVN INMKKDDEES EDK *length of your protein in Amino Acids 633 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 70129.9 Molar Extinction coefficient of your protein at 280 nm wavelength: 22015 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only): *GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
ATP-dependent zinc metalloprotease/ FtsH
*NCBI Gene # or RefSeq#: 1392
*Protein ID (NP or XP #) or Wolbachia#:
*Organism (including strain): Bacillus Anthracis
Etiologic Risk Group (see link below): Risk Group Lv 3 (high individual risk, low community risk)
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Anthrax is a serious infectious disease caused by gram-positive, rod-shaped bacteria known as Bacillus anthracis. Anthrax can be found naturally in soil and commonly affects domestic and wild animals around the world.
The bacterial cell lives as a hardy spore to survive harsh conditions. The spores germinate into thriving colonies of bacteria once inside an animal or person. Anthrax usually affects livestock far more than humans, but—as we know from the 2001 anthrax attacks in the United States—anthrax is feared as a modern biological weapon.
Anthrax occurs in three forms:
1.Cutaneous (affecting the skin)
2.Inhalational (in the lungs)
Gastrointestinal (in the digestive tract)
Link to TDR Targets page (if present):
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
http://www.ncbi.nlm.nih.gov/gene/1086600
Essentiality of this protein:
Targets mitogen-activated protein kinase cleaving them to remove a small N-terminal stretch but leaving the bulk of the protein, including the protein kinase domain, intact. LF-mediated cleavage of MEK1 and MKK6 has been shown to inhibit signalling through their cognate MAPK pathways
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): multimer
Complex of proteins?:
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
*EC#: 3.4.24-
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/search_result.php?a=13&RN=&RNV=1&os=1&pt=&FNV=1&tt=&SYN=&Textmining=&T[0]=2&T[1]=2&V[1]=1&V[2]=2&W[3]=metalloprotease&T[3]=2&l=10,10#results
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives
---- Chain used for homology:
Current Inhibitors:
tissue inhibitors of metalloproteinases (TIMP1,2,3,4)
Expression Information (has it been expressed in bacterial cells): E.coli
Purification Method:
Amino acids 147–610 of FtsH from T. maritima were amplified from genomic DNA and ligated into a pET28a expression vector (Stratagene) adding a C-terminal hexa-His-tag. Expression in Rosetta (DE3) cells (Novagen) was induced by adding 0.5 mM IPTG at an OD600 of 0.5. Heat precipitation of E. coli proteins was carried out at 75°C for 3 min. The supernatant was further purified by Ni-NTA and anion-exchange chromatography. (Δtm)FtsH-containing fractions were again pooled and loaded onto an S75 size-exclusion column (Amersham Pharmacia Biosciences) equilibrated with buffer G (100 mM NaCl/2 mM DTT/20 mM Tris, pH 8.0/0.02% sodium azide).
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1413944/
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MNRIFRNTIF YLLIFLVVIG IVSYFNGSTQ KTTSVSYDKF ITKLEKGEVR
NVQLQPKNGV FEVKGQFNNS SQGEQFVTYA PNTEELQKKI NDKAQGAEVK
YQPAEETSAW VTFFTSIIPF VIIFILFFFL LNQAQGGGSR VMNFGKSKAK
LYNDEKKKVR FRDVAGADEE KQELVEVVEF LKDPRKFAEV GARIPKGVLL
VGPPGTGKTL LARAVAGEAG VPFFSISGSD FVEMFVGVGA SRVRDLFENA
KKNAPCIIFI DEIDAVGRQR GAGLGGGHDE REQTLNQLLV EMDGFGANEG
IIIIAATNRP DILDPALLRP GRFDRQITVD RPDVNGREAV LKVHARNKPL
DENINLRAIA TRTPGFSGAD LENLLNEAAL VAARQDKKKI DMSDIDEATD
RVIAGPAKKS RVISEKERNI VAFHEAGHTV IGVVLDEADV VHKVTIVPRG
QAGGYAVMLP KEDRYFMTKP ELLDKITGLL GGRVAEEIVF GEVSTGAHND
FQRATGIARR MVTEFGMSDK LGPMQFGSSQ GGQVFLGRDF HSEQNYSDAI
AHEIDMEMQT IMKECYARAK QILTDNRDKL DLIAKTLLEV ETLDAEQINH
LCDYGRLPER PTSSADVKVN INMKKDDEES EDK
*length of your protein in Amino Acids 633
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 70129.9
Molar Extinction coefficient of your protein at 280 nm wavelength: 22015
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**